Use of soluble st2 in the prediction of hormone-resistant icis-associated myocarditis

Abstract

The application discloses application of sST2 alone or sST2 in combination with cTnT and CK-MB as a biomarker in preparation of a srICIAM diagnosis or prediction reagent for hormone-resistant ICIs-related myocarditis. When the serum sST2 concentration of a to-be-detected donor is greater than or equal to 36.96 ng / mL, it is indicated that the patient is a high-risk population of srICIAM; when the serum sST2 concentration is greater than or equal to 36.96 ng / mL and the cTnT concentration is greater than or equal to 511.0 pg / mL, the patient is combined with severe myocardial injury, and the risk of developing hormone-resistant ICIs-related myocarditis is significantly increased. The application realizes early warning, risk stratification and accurate prediction of srICIAM based on a noninvasive detection scheme of peripheral blood markers, and provides a brand-new technical strategy for noninvasive in vitro diagnosis and early clinical intervention of srICIAM.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology and relates to the use of soluble growth-stimulating gene 2 protein (sST2) combined with cardiac troponin T (cTnT) as a biomarker to predict the risk of steroid-resistant immune checkpoint inhibitor-associated myocarditis (srICIAM) in patients with ICIs-associated myocarditis. It can also be further combined with creatine kinase isoenzyme (CK-MB) to achieve joint detection and assessment. Background Technology

[0002] In recent years, immunotherapy, primarily based on immune checkpoint inhibitors (ICIs), has revolutionized cancer treatment, ushering in a new era of cancer immunotherapy. However, activation of the immune system can induce immune-related adverse effects (irAEs). ICI-associated myocarditis is one of the less common but most serious irAEs, with a mortality rate as high as 46%.

[0003] Glucocorticoids are the first-line treatment for ICIs-related myocarditis. The 2022 European Society of Cardiology guidelines recommend that once ICIs-related myocarditis is suspected, high-dose glucocorticoid pulse therapy should be initiated as early as possible to reduce major adverse cardiovascular events (MACE) and mortality. For patients who do not experience a significant decrease in cardiac troponin T (cTnT) (a decrease of less than 50%) after 3 days of adequate intravenous methylprednisolone treatment, or who still have atrioventricular block, ventricular arrhythmias, or left ventricular dysfunction, the 2022 European Society of Cardiology defines these patients as hormone-resistant ICIs-related myocarditis (srICIAM).

[0004] In patients with hormone-resistant ICIs, second-line immunosuppressive drugs are added to glucocorticoid therapy, including mycophenolate mofetil, intravenous immunoglobulin, plasma exchange, tocilizumab, and tofacitinib. However, regardless of the second-line immunosuppressive drug added, the mortality rate of patients with hormone-resistant ICIs-related myocarditis remains as high as 50%. ICIs-related myocarditis has a rapid onset and progression. Early assessment of the prognosis of patients with ICIs-related myocarditis and timely implementation of appropriate treatment measures can reduce mortality to some extent. Therefore, how to effectively identify srICIAM and predict its prognosis has become a key research focus that urgently needs to be addressed in clinical practice. Currently, there is still a lack of a testing protocol that is simple to use, accurate, rapid, efficient, minimally invasive, and reliable for the early prediction and prognostic assessment of hormone-resistant ICIs-related myocarditis. Clinically, there is an urgent need to establish a precise and convenient method for risk prediction of hormone-resistant ICIs-related myocarditis. Summary of the Invention

[0005] Addressing the clinical challenge of unpredictable risk in hormone-resistant ICIs-associated myocarditis (srICIAM), the inventors conducted a study based on blood samples collected in recent years from cancer patients receiving immune checkpoint inhibitor therapy and diagnosed with ICIs-associated myocarditis (including patients who had received glucocorticoid therapy, such as methylprednisolone). Differentially expressed proteins associated with myocarditis were screened and analyzed. The study found a statistically significant difference in the expression level of growth-stimulating gene 2 (sST2) protein between hormone-resistant (srICIAM) and hormone-sensitive (Non-srICIAM) patients. Serum sST2 levels in srICIAM patients were significantly higher than those in hormone-sensitive patients. Simultaneously, myocarditis markers such as cTnT, cTnIplus, CK-MB, and MYO also showed differential expression between hormone-resistant and hormone-sensitive patients. The above results suggest that sST2 can be used alone as a biomarker for srICIAM. Combining sST2 with cardiac troponin T (cTnT) and creatine kinase isoenzyme MB (CK-MB) can further improve predictive efficacy for predicting the risk of developing hormone-resistant subtypes in patients with ICIs-related myocarditis. Based on the above research results, this invention provides the following technical solution:

[0006] This invention mainly provides a biomarker application in the preparation of diagnostic / risk prediction reagents for hormone-resistant ICIs-associated myocarditis (srICIAM); the biomarker is soluble growth-stimulating gene 2 protein, i.e., sST2, or the biomarker is a combination of sST2 and cardiac troponin T, i.e., cTnT, sST2+cTnT, a combination of sST2 and creatine kinase isoenzyme MB, i.e., CK-MB, sST2+CK-MB, or a combination of sST2 and cTnT and CK-MB, sST2+cTnT+CK-MB.

[0007] In one specific implementation, srICIAM diagnosis / risk prediction is performed by detecting the expression level of sST2 in the biological sample of the donor patient; or simultaneously detecting the expression levels of sST2 and CK-MB; or simultaneously detecting the expression levels of sST2, CK-MB and cTnT. The biological sample is selected from the following group: whole blood, plasma, serum, tumor tissue sample, tissue fluid, saliva, oral mucosa, nasopharyngeal secretions, body fluid, and urine. Preferably, the biological sample is whole blood, plasma or serum, more preferably serum. This is adapted to the marker threshold interpretation system defined in this invention, and the detection results have high accuracy.

[0008] Optionally, the expression level of sST2 in the donor patient's biological sample can be determined by gene / mRNA detection or protein detection.

[0009] Preferably, the biological sample is serum. The diagnostic / risk prediction criteria for srICIAM are as follows: when the sST2 level / concentration in the donor serum is above 36.96 ng / mL, it indicates that the donor is in a high-risk group for srICIAM; or

[0010] When the serum sST2 level / concentration of the donor is ≥ 36.96 ng / mL and the serum cTnT level / concentration is ≥ 511.0 pg / mL, it indicates that the donor patient has severe myocardial injury and a high risk of developing hormone-resistant ICIs-related myocarditis.

[0011] In one specific embodiment of the present invention, the above-mentioned srICIAM hierarchical interpretation criteria are constructed into a computable algorithm model, which can be imported into a detection system such as a protein detection device equipped with a computer or networked to achieve automated risk assessment. The detection system specifically includes an analysis unit and an evaluation unit, wherein the analysis unit is used to detect the expression concentration levels of sST2, cTnT, and CK-MB target proteins in serum biological samples, and the evaluation unit can automatically complete the risk assessment, severity scoring, and prognosis judgment of hormone-resistant ICIs-related myocarditis based on the protein detection results output by the analysis unit and in combination with the aforementioned hierarchical interpretation algorithm model.

[0012] Optionally, the above reagents are used to detect the content of sST2, cTnT and CK-MB proteins in biological samples based on the following principles: ELISA (enzyme-linked immunosorbent assay), chemiluminescent immunoassay, flow cytometry microsphere detection, Olink proximity extension technology, multiple inflammatory protein detection technology, and Luminex multiplex factor detection technology.

[0013] In one application of the present invention, the reagent is a kit, namely an sST2 detection kit for diagnosing hormone-resistant ICIs-related myocarditis; or an sST2+cTnT and / or CK-MB detection kit for diagnosing hormone-resistant ICIs-related myocarditis.

[0014] Preferably, the kit is a protein detection reagent based on the principle of antibody-antigen affinity binding.

[0015] In the above kit, the antibody used to recognize sST2 is selected from sST2 monoclonal antibody or sST2 polyclonal antibody; the antibody used to recognize cTnT is selected from cTnT monoclonal antibody or cTnT polyclonal antibody; and the antibody used to recognize CK-MB is selected from CK-MB monoclonal antibody or CK-MB polyclonal antibody.

[0016] In one specific embodiment, the reagent is a kit selected from latex immunoturbidimetric assay kits, magnetic microparticle chemiluminescence assay kits, radioimmunoassay kits, or immunochromatographic test strips.

[0017] Optionally, the above kit is a double-antibody sandwich kit, selected from the following group of methods:

[0018] A. Enzyme-linked immunosorbent assay kit;

[0019] B. Magnetic microparticle chemiluminescence assay kit;

[0020] C. Immunochromatographic test strips.

[0021] Preferably, any of the above-described reagent kits of the present invention further includes an instruction manual.

[0022] The format of the instruction manual is not strictly limited. It can be printed on the reagent bottles, test tubes and similar items, or plates that come with the reagent kit. It can also be a separate paper document that can be posted or stored on the outside or inside the reagent kit container. For example, it can be a paper document with an operation demonstration video app download window or a QR code. The instruction manual can also be in multimedia format, such as a CD, USB flash drive, or cloud storage.

[0023] This invention is the first to clearly demonstrate that sST2 can serve as an independent biomarker for indicating and warning of hormone-resistant ICIs-related myocarditis (srICIAM). It can also be used in combination with cTnT and CK-MB, specific biomarkers for myocarditis, to achieve accurate risk prediction of srICIAM and precise differential diagnosis of severe myocardial injury. Clinically, by detecting sST2 expression levels in patients in conjunction with cTnT, and by performing synergistic analysis with cTnT and CK-MB biomarkers, early and accurate prediction, risk stratification, and prognostic assessment of srICIAM can be achieved. The detection method of this invention is simple to operate, rapid, and reliable, effectively identifying high-risk patients with hormone resistance in ICIs-related myocarditis. It provides precise laboratory evidence for early clinical intervention and individualized treatment planning, effectively improving patient clinical prognosis and reducing mortality from severe cases, demonstrating significant clinical application value and promising prospects for widespread application. Attached Figure Description

[0024] Figure 1 ROC curves of independent and combined indicators of sST2 combined with cTnT and CK-MB for predicting hormone-resistant ICIs-related myocarditis. Detailed Implementation

[0025] In our years-long research on the clinical diagnosis and treatment of immune checkpoint inhibitor-associated myocarditis (ICIAM), our research group has isolated and examined numerous proteins and gene mRNAs highly expressed in patients with several types of ICIAM from blood samples, hoping to discover biomarkers that can be used for the diagnosis of various types of ICIAM. For example, the patent document with publication number CN121253827A reports that the combination of CXCL9, MMP1, and TRANCE can be used to predict hormone-resistant immune checkpoint inhibitor-associated myocarditis (srICIAM). The discovery of protein or gene biomarkers that can be used alone to assess the risk level and even the severity of srICIAM would undoubtedly make srICIAM prediction more convenient.

[0026] In our recent study, we found that growth stimulation expressed gene 2 (ST2), especially soluble growth stimulation expressed gene 2 (sST2), has a clear indicative effect on srICIAM prediction, suggesting that it can be used alone as a biomarker for srICIAM.

[0027] Growth-stimulated gene 2 (ST2) protein is a member of the interleukin-1 (IL-1) receptor superfamily, primarily expressed on fibroblasts and immune cells. It exists in two forms: a membrane-bound receptor (ST2L) and a soluble ST2, sST2. ST2 is known to form a signaling pathway with IL-33, and this pathway plays a crucial role in various cardiovascular diseases and tumor immune responses. The ST2 / IL-33 signaling pathway exerts a protective effect in atherosclerosis by inhibiting inflammatory responses. Furthermore, ST2 / IL-33 also participates in regulating T cell-mediated immune responses. It is generally believed that sST2, acting as a decoy receptor, preferentially binds to free IL-33, thereby inhibiting ST2 / IL-33 signaling.

[0028] Studies have shown that elevated serum sST2 levels are associated with mechanical stress or inflammation. When the heart is subjected to strong mechanical stress or severe inflammatory stimulation, cardiomyocytes are damaged, and sST2 can be released from the damaged myocardium into the blood, further increasing the damaging effect on the heart. Recent studies have shown that sST2 has potential diagnostic and prognostic value for pathological states such as fulminant myocarditis, heart failure, and myocardial infarction. However, the potential of sST2 as a biomarker for hormone-resistant ICIs-related myocarditis remains to be explored. This invention explores the predictive value of sST2 combined with the traditional biomarker cTnT for the prognosis of patients with ICIs-related myocarditis, especially those with hormone-resistant ICIAM (srICIAM).

[0029] Furthermore, the combination of sST2 with cardiac troponin T (cTnT) and / or creatine kinase isoenzyme MB (CK-MB), sST2+cTnT / CK-MB, not only helps improve the predictive accuracy of srICIAM, but can also determine whether a patient has myocardial injury.

[0030] In the description of the technical solutions of this invention, the term "and / or" used in terms such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0031] Detecting the content / level / concentration of a specific protein in biological samples, especially blood samples, is a mature technology, often based on the antibody-antigen affinity binding principle. This is easily achieved using ELISA, and such kits are widely used in clinical diagnostics. Therefore, when detecting sST2 and cTnT / CK-MB levels using ELISA, kits can be easily developed. sST2, cTnT, and CK-MB can be detected together in the same kit or separately in different kits. This srICIAM kit includes at least:

[0032] (1) Antibodies that specifically bind to sST2 / cTnT / CK-MB proteins, which may also be referred to as sST2 / cTnT / CK-MB primary antibodies in this article; and

[0033] (2) A standard sample consisting of a solution containing a known amount of sST2 / cTnT / CK-MB, wherein sST2 / cTnT / CK-MB may be a natural sST2 / cTnT / CK-MB protein extracted from humans or mammals or a recombinant sST2 / cTnT / CK-MB protein produced by fermentation of genetically engineered bacteria.

[0034] In this case, the kit can detect sST2 / cTnT / CK-MB using a latex immunoturbidimetric assay with an sST2 / cTnT / CK-MB antibody, a chemiluminescence assay such as magnetic microparticle chemiluminescence, or a radioimmunoassay (RIA) using a radiolabeled sST2 / cTnT / CK-MB antibody. That is, the kit can be a latex immunoturbidimetric assay kit, a magnetic microparticle chemiluminescence assay kit, a radioimmunoassay kit, or an immunochromatographic test strip.

[0035] Optionally, the above-mentioned srICIAM detection kit may further include: (3) a labeled secondary antibody, wherein the labeled secondary antibody is a second antibody that can specifically bind to the sST2 / cTnT / CK-MB antigen complex captured by the first antibody; the second antibody carries a label that can catalyze or activate a chemiluminescent compound or fluorescent dye, thereby rapidly converting a colorless substrate into a colored product, or causing a light change, or converting a non-fluorescent fluorescent dye into a strongly fluorescent product, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase); or the label on the second antibody is a chemiluminescent compound or fluorescent dye that can be catalyzed or activated to produce color or light, such as acridinium ester.

[0036] The kit of this invention can employ a double-antibody sandwich detection system, containing two specific antibodies against sST2 / cTnT / CK-MB: (1) is a capture antibody (primary antibody), used to capture sST2 / cTnT / CK-MB antigen in the sample; (3) is a detection antibody (secondary antibody), used to specifically identify the captured antigen. The two antibodies correspond to different epitopes of the antigen protein. First, the primary antibody binds to the antigen to form a primary antibody-antigen complex, and then the secondary antibody binds to the complex to form a primary antibody-antigen-second antibody sandwich structure, thereby achieving specific quantitative detection of sST2 / cTnT / CK-MB protein.

[0037] The srICIAM detection kit described in this invention is preferably a double-antibody sandwich assay kit, specifically selectable from the following types: A. Enzyme-linked immunosorbent assay (ELISA) kit; B. Magnetic microparticle chemiluminescence assay kit; C. Immunochromatographic test strip. The first and second antibodies used in this invention are not limited to specific antibody types; monoclonal antibodies or polyclonal antibodies can be used, and they can be flexibly combined according to the detection scenario. An exemplary combination is: the first antibody is a monoclonal antibody, and the second antibody is a polyclonal antibody, which can balance antigen binding specificity and detection sensitivity.

[0038] Optionally, the aforementioned sST2 / cTnT / CK-MB primary antibody can be a primary antibody labeled with quantum dots or fluorescent microspheres, or it can be labeled with colloidal gold. Quantum dots are nanoparticles composed of semiconductor materials such as cadmium selenide (CdSe). When excited by ordinary light, quantum dots can emit fluorescence that is tens of times stronger than that of ordinary fluorescein, and they have a broad excitation spectrum and a narrow emission spectrum. The emission spectrum can also be adjusted according to the different semiconductor materials and the different sizes of nanoparticles, and the emitted light is highly stable and will not be quenched. Due to these advantages of quantum dot fluorescence, antibodies labeled with quantum dot nanoparticles can have a luminescence intensity hundreds of times higher than those labeled with ordinary fluorescein, thereby enabling the signal sensitivity of the reagent to reach a level comparable to chemiluminescence.

[0039] sST2 / cTnT / CK-MB monoclonal antibodies and sST2 / cTnT / CK-MB polyclonal antibodies can be commercially available antibodies or prepared using conventional techniques, such as using sST2 / cTnT / CK-MB or preferably human sST2 / cTnT / CK-MB as antigens.

[0040] Preferably, when the above-mentioned kit is an ELISA kit, it further comprises:

[0041] (4) Initiator solution, used to initiate a chemical reaction between the marker on the sST2 / cTnT / CK-MB secondary antibody and the chemiluminescent compound, chromogenic substrate or fluorescent dye that is the target of the marker reaction.

[0042] When the above kit is an ELISA kit, it is preferable to use the sST2 / cTnT / CK-MB primary antibody, or a solid-phase support for immobilizing the sST2 / cTnT / CK-MB primary antibody, such as a multi-well plate or magnetic microparticles, with a chemiluminescent compound or fluorescent dye attached. This simplifies the immunoassay method and improves quantification accuracy.

[0043] Preferably, the biological sample may be selected from the group consisting of whole blood, plasma, serum, saliva, oral mucosa, nasopharyngeal secretions, tissue fluid, body fluid, and urine, such as serum.

[0044] Preferably, any of the above-mentioned reagent kits also includes an instruction manual.

[0045] To achieve simplicity and rapid practicality, the detection of sST2, cTnT, and CK-MB can be performed using a kit. Those skilled in the art will readily understand that, in addition to commonly used biological and chemical reagents for detecting sST2, cTnT, and CK-MB, the kit of this invention may also include at least one of the following: a carrying tool, the space of which is divided into defined spaces capable of accommodating one or more containers, 96-well plates, or strips, such as kits, vials, test tubes, and the like, each containing a single component for the method of this invention; and an instruction manual, which may be written on the vials, test tubes, and the like, or on a separate piece of paper, or on the outside or inside of the container, for example, a paper document with an operation demonstration video / APP download window such as a QR code. The instruction manual may also be in a multimedia tangible or intangible form, such as a USB flash drive or cloud storage.

[0046] Optionally, the instruction manual may also include a standard reference table for distinguishing between srICIAM and srICIAM+ myocardial injury, which records cutoff values ​​or thresholds for the concentrations of sST2 / cTnT / CK-MB (e.g., serum levels of sST2 / cTnT / CK-MB) in blood samples that distinguish srICIAM.

[0047] Since serum is the most commonly used test sample in hospitals, and the expression levels of sST2, cTnT, and CK-MB can all be determined by serum testing, early risk prediction of hormone-resistant ICIs-related myocarditis can be achieved by non-invasive peripheral blood testing. This approach balances the convenience of testing with the accuracy of prediction, and solves the technical difficulties of existing technologies in accurately predicting hormone-resistant subtypes of ICIs myocarditis and the poor efficacy of prognostic assessment.

[0048] In one srICIAM prediction method, this invention prioritizes the use of a combined sST2+cTnT prediction model to stratify patient risk. By jointly determining these two indicators, it differentiates between hormone-sensitive ICIs-related myocarditis and the hormone-resistant high-risk subtype. For patients identified as high-risk for hormone-resistant ICIs-related myocarditis by this invention, clinical intervention can be initiated earlier than expected, without waiting for the onset of severe and refractory symptoms. This allows for early and precise intervention, shortening treatment lag time and reducing mortality from severe illness.

[0049] Compared to existing srICIAM diagnostic and assessment technologies, this invention possesses significant innovation and technological advantages, as detailed below:

[0050] 1. Breaking through the limitations of traditional biomarker mechanisms, this invention achieves specific identification of hormone-resistant ICIs-related myocarditis. Current clinically routine use of myocardial biomarkers such as cTnT and CK-MB only reflects acute myocardial necrosis and injury, failing to reflect the persistent immune inflammatory activation and myocardial remodeling state mediated by ICIs, and lacking the ability to differentiate between hormone-sensitive and hormone-resistant ICIs-related myocarditis. This invention is the first to apply sST2 to the prediction of hormone-resistant ICIs-related myocarditis. Utilizing the biological characteristics of sST2, which specifically reflects the body's immune inflammatory burden, myocardial inflammatory damage, and adverse remodeling, it can accurately identify high-risk patients who are unresponsive to glucocorticoid therapy and require second-line immunosuppressive salvage therapy, overcoming the difficulty of precise disease subtype stratification in existing technologies.

[0051] 2. Constructing a complementary joint prediction model significantly improves prediction accuracy, balancing sensitivity and specificity, and substantially enhancing predictive efficacy. Traditional single biomarkers generally suffer from efficacy limitations: cTnT has high specificity but insufficient sensitivity for inflammation stratification and a high rate of missed diagnoses in high-risk cases; sST2 alone has limited predictive ability. This invention, based on the complementary mechanism principle of inflammatory remodeling and myocardial necrosis, constructs a joint prediction model of sST2 and traditional myocardial biomarkers. The cTnT+sST2 joint model achieves an AUC of 0.857 and a specificity of 100.0%, while significantly improving sensitivity. This completely solves the technical challenge of simultaneously achieving high sensitivity and specificity with a single indicator, and its predictive performance is significantly superior to existing single detection methods.

[0052] Example

[0053] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the invention.

[0054] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the mass percentage.

[0055] The commercial reagent kits used in the examples were operated according to their instructions.

[0056] Example 1: Predicting hormone-resistant ICI-related myocarditis by serum soluble ST2 levels

[0057] 1. Test method:

[0058] 1.1 Research Subjects

[0059] Patients who received ICIs treatment at Zhongshan Hospital affiliated with Fudan University between March 2023 and February 2024 and were diagnosed with ICIs-related myocarditis through a multidisciplinary outpatient consultation at the cardiology or oncology department were selected. The diagnosis of ICIs-related myocarditis was based on the Bonaca criteria. All patients with ICIs-related myocarditis received standardized drug treatment during hospitalization and after discharge.

[0060] Based on the dynamic changes in cTnT after glucocorticoid therapy, patients with ICIs-related myocarditis are classified into hormone-resistant and hormone-sensitive types. The diagnosis of hormone-resistant ICIs-related myocarditis follows the 2022 European Society of Cardiology guidelines, defined as: no significant decrease in cTnT (less than 50%) after 3 days of adequate intravenous methylprednisolone treatment, or the presence of atrioventricular block, ventricular arrhythmias, or left ventricular dysfunction. Hormone-resistant patients require additional immunosuppressive drugs in addition to glucocorticoid therapy.

[0061] 1.2 Sample Collection and Testing

[0062] Peripheral blood was collected from patients before they received glucocorticoid therapy. After standing at 4°C for 30 min, the blood was centrifuged at 3500 rpm for 10 min (4°C) to separate the supernatant. The supernatant was aliquoted into 1 ml EP tubes and stored at -80°C. Blood biochemistry was performed using Baditai Isucid. The AFIAS-6 fully automated fluorescence immunoassay analyzer uses a dry immunofluorescence quantification method to detect sST2. The sST2, cTnT, cTnI plus / CK-MB / MYO kits were all provided by Badite (Guangxi) Biotechnology Co., Ltd. Specific testing procedures were strictly followed according to the kit instructions.

[0063] Methodological quality control: Instrument calibration is completed before daily testing, and internal quality control is performed using the quality control materials provided with the reagent kits to ensure that the quality control results are within the manufacturer's allowable range before clinical samples can be tested; all samples are tested in the same batch, and the results are independently verified by two technicians to ensure that the data are accurate and reliable.

[0064] The cutoff value for sST2 testing is 35 ng / mL, with a detection range of 3.1-200 ng / mL. A positive result is considered to be between 35-70 ng / mL, and a result >70 ng / mL is considered strongly positive, indicating a significantly increased incidence of adverse events requiring close monitoring. The cutoff value for CK-MB testing is 3 ng / mL, with a detection range of 3-100 ng / mL. A positive result is considered to be between 3-7 ng / mL, and a result >7 ng / mL indicates myocardial injury. The cutoff value for cTnT testing is 17 pg / mL, and a result of 17-100 pg / mL indicates myocardial injury. The cutoff value for Tn-I plus testing is 0.04 ng / mL, and a result of 0.04-0.3 ng / mL indicates myocardial injury. The cutoff value for MYO testing is 2 ng / mL, with a low risk of 2-70 ng / mL and a high risk of >70 ng / mL; the detection range is 0-500 ng / mL.

[0065] 1.3 Data Collection

[0066] Demographic data (sex and age), history of underlying diseases (hypertension, coronary artery disease, diabetes, hyperlipidemia, atrial fibrillation, etc.), type of primary tumor, history of ICI and other anti-tumor drug treatments, time from initiation of ICI treatment to enrollment, imaging examinations such as electrocardiogram, echocardiography, cardiac MRI, laboratory tests such as myocardial injury markers (cTnT, CK, CK-MB, NT-proBNP), liver function (ALT, AST), and other irAEs were collected. irAEs were defined and graded according to the National Cancer Institute Adverse Events Criteria (CTCAE 4.0), and the type, grade, time, treatment, and outcome of irAEs were recorded. Follow-up was conducted at 3 months and 6 months, with the primary endpoint being all-cause mortality.

[0067] 1.4 Statistical Methods

[0068] Data analysis was performed using SPSS 26.0 (IBM SPSS Statistics). Graphpad Prism 9 for macOS (GraphPad Software, LLC) and R software (version 4.5.1; R Foundation for Statistical Computing, Vienna, Austria) were used for graph plotting. Continuous variables were expressed as mean ± SD or Median (IQR Q1-Q3). The Shapiro-Wilk test was used to determine whether the data followed a normal distribution. If the data followed a normal distribution, the independent samples t-test was used for comparisons between groups; if the data did not follow a normal distribution, the Mann-Whitney U test was used for comparisons between groups. Categorical variables were expressed as n (%), and comparisons between groups were performed using the χ² test or Fisher's exact test. The area under the ROC curve was used to evaluate the value of baseline sST2, CK-MB, and other myocardial injury markers in ICIs-related myocarditis and hormone-resistant ICIs-related myocarditis. P < 0.05 was considered statistically significant.

[0069] 2. Experimental Results

[0070] 2.1 Patient Baseline Characteristics

[0071] This study included 28 patients with ICIs-related myocarditis. Based on follow-up results, 15 patients (53.6%) had hormone-resistant ICIs-related myocarditis (srICIAM group), and 13 patients had hormone-sensitive ICIs-related myocarditis (Non-srICIAM group). The average age of patients in the srICIAM group was (67.2 ± 6.4) years, and the average age of patients in the hormone-sensitive group was (68.0 ± 11.7) years; there was no statistically significant difference between the two groups (P>0.05). Ten patients (66.7%) in the srICIAM group and 11 patients (84.6%) in the non-srICIAM group were male; there was no statistically significant difference between the two groups (P>0.05). There were no statistically significant differences between the two groups in terms of past cardiovascular history, primary tumor type, type of immunotherapy received, and combined antitumor therapy (P>0.05) (Table 1).

[0072] Table 1. Patient baseline characteristics

[0073] Index srICIAM cohort (n=15) Non-srICIAM cohort (n=13) P Age / year, mean ± SD 67.2 ± 6.4 68.0 ± 11.7 0.805 Sex Male,n (%) 10 (66.7) 11 (84.6) 0.267 Female, n (%) 5(33.3) 2 (15.4) Underlying conditions Hypertension, n (%) 5 (33.3) 7 (53.8) 0.274 Hyperlipidemia, n (%) 1 (6.7) 0 0.258 Diabetes, n (%) 2 (13.3) 4 (30.8) 0.260 Indication of ICI Lung cancer, n (%) 4 (26.7) 5 (38.5) 0.505 Gastrointestinal cancer, n (%) 5 (33.3) 3 (23.1) 0.547 Hepatocellular cancer, n (%) 3 (20.0) 1 (7.7) 0.342 Nasopharyngeal malignancy, n (%) 0 (0) 2 (15.4) 0.072 Renal cancer, n (%) 0 1 (7.7) 0.209 Bladder cancer, n (%) 2 (13.3) 0 0.105 Others, n (%) 1 (6.7) 1 (7.7) 0.916 Type of immune therapy Sintilimab, n (%) 5 (33.3) 2 (15.4) 0.267 Pembrolizumab, n (%) 1 (6.7) 3 (23.1) 0.210 Tislelizumab, n (%) 2 (13.3) 3 (23.1) 0.502 Camrelizumab, n (%) 1 (6.7) 1 (7.7) 0.916 Toripalimab, n (%) 1 (6.7) 1 (7.7) 0.916 Atezolizumab, n (%) 1 (6.7) 1 (7.7) 0.916 Durvalumab, n (%) 2 (13.3) 0 0.105 Penpulimab, n (%) 1(7.7) 0 0.209 Cadonilimab, n (%) 0 1 (7.7) 0.209 Data unspecified, n (%) 2 (13.3) 0 0.105 Type of ICI PD-1, n (%) 10 (66.7) 11 (84.6) 0.267 PD-L1, n (%) 3 (20.0) 1 (7.7) 0.342 PD-1+CTLA-4, n (%) 0 1(7.7) 0.209 Anti-tumor drugs 0.124 ICI monotherapy, n (%) 1 (6.7) 2 (15.4) ICI plus chemotherapy, n (%) 11 (73.3) 5 (38.5) ICI plus molecularly targeted agents, n (%) 2 (13.3) 1(7.7) ICI plus molecularly targeted agents and chemotherapy, n (%) 1(6.7) 5 (38.5)

[0074] In the table, ICI stands for immune checkpoint inhibitor; srICIAM stands for steroid-resistant immune checkpoint inhibitor-associated myocarditis; PD-1 stands for programmed cell death-protein 1; PD-L1 stands for programmed cell death ligand 1; and CTLA-4 stands for cytotoxic T-lymphocyte-associated protein 4.

[0075] 2.2 Comparison of baseline serum sST2, cTnT, CK-MB, cTnI, and MYO levels between the two groups of patients

[0076] Compared with the hormone-sensitive group, patients in the srICIAM group had significantly higher baseline levels of sST2, cTnT, and CK-MB (P<0.05). There were no significant differences in cTnI and MYO levels between the srICIAM group and the Non-srICIAM group (P>0.05) (Table 2).

[0077] Table 2. Comparison of serum sST2, cTnT, CK-MB, cTnI, and MYO levels between the srICIAM group and the Non-srICIAM group.

[0078] Biomarker, median (IQR) srICIAM cohort (n=15) Non-srICIAM cohort (n=13) P sST2(ng / mL) 59.15 (17.29, 103.16) 21.09 (12.23,33.36) 0.037 cTnT (pg / mL) 498.38 (244.2, 708.16) 127.55 (66.75, 415.02) 0.011 CK-MB (ng / mL) 61.06 (22.41, 100.0) 12.61 (8.92, 59.14) 0.012 cTnI (ng / mL) 0.15 (0.035, 6.323) 0.03 (0.01, 0.44) 0.094 MYO (ng / mL) 474.62 (238.82, 500.00) 162.94 (78.53, 470.75) 0.085

[0079] 2.3 ROC curve analysis of the predictive value of sST2, cTnT, and CK-MB for srICIAM

[0080] This study statistically analyzed the predictive value of sST2, cTnT, and CK-MB for srICIAM (Table 3). Independent index ROC curves (see Table 3). Figure 1The results showed that when the sST2 peak value was taken at the maximum Youden index of 0.446, the optimal cutoff value was 36.96 ng / ml, with a sensitivity of 60.0%, a specificity of 84.6%, and an area under the curve (AUC) of 0.733 (95% CI: 0.544–0.922, P = 0.036). When the cTnT peak value was taken at the maximum Youden index of 0.500, the optimal cutoff value was 511.0 pg / ml, with a sensitivity of 50.0%, a specificity of 100%, and an AUC of 0.786 (95% CI: 0.614–0.957, P = 0.012). When the peak value of CK-MB is taken as the maximum value of Youden index of 0.544, the optimal cutoff value is 14.27 ng / ml, the sensitivity is 92.9%, the specificity is 61.5%, and the area under the curve (AUC) is 0.780 (95% CI: 0.601-0.959, P = 0.013) (Table 3).

[0081] ROC curve analysis of the combined sST2, cTnT, and CK-MB indices showed that the predictive performance was significantly improved compared to individual indices when the indices were combined. The combination of cTnT and sST2 (hereinafter referred to as cTnT+sST2) yielded an AUC of 0.857 for predicting srICIAM, a model sensitivity of 71.4%, a specificity of 100.0%, and a maximum Youden index of 0.714 (Table 3). The combined CK-MB and sST2 indices showed an AUC of 0.813 for predicting srICIAM, a model sensitivity of 78.6%, a specificity of 76.9%, and a maximum Youden index of 0.555.

[0082] Table 3. ROC analysis results of serum sST2, cTnT, and CK-MB in predicting srICIAM

[0083] Cut-off value Sensitivity Specificity Youden Index AUC 95% CI P sST2(ng / ml) 36.96 0.600 0.846 0.446 0.733 0.544-0.922 0.036 cTnT( pg / ml) 511.0 0.500 1.000 0.500 0.786 0.614-0.957 0.012 CK-MB (ng / ml) 14.27 0.929 0.615 0.544 0.780 0.601-0.959 0.013 sST2+cTnT - 0.714 1.000 0.714 0.857 0.712-1.000 0.002 sST2+CK-MB - 0.786 0.769 0.555 0.813 0.651-0.975 0.006 sST2+cTnT+CK-MB - 0.714 1.000 0.714 0.857 0.712-1.000 0.002

[0084] 3. Experimental Conclusions

[0085] sST2 has certain predictive value for srICIAM. Compared with a single indicator, sST2 combined with cTnT significantly improves diagnostic efficacy and helps clinicians identify high-risk patients and optimize treatment strategies.

[0086] It should be understood that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a biomarker in the preparation of a diagnostic / risk prediction reagent for hormone-resistant ICIs-associated myocarditis (srICIAM); the biomarker is soluble growth-stimulating gene 2 protein, i.e., sST2, or the biomarker is a combination of sST2 and cardiac troponin T, i.e., cTnT, sST2+cTnT; a combination of sST2 and creatine kinase isoenzyme MB, i.e., CK-MB, sST2+CK-MB; or a combination of sST2 and cTnT and CK-MB, sST2+cTnT+CK-MB.

2. The application of biomarkers as described in claim 1, characterized in that, srICIAM diagnosis / risk prediction is performed by detecting the expression level of sST2 in the donor's biological sample; or simultaneously detecting the expression levels of sST2 and CK-MB; or simultaneously detecting the expression levels of sST2, CK-MB and cTnT. The biological sample is selected from the group consisting of whole blood, plasma, serum, tumor tissue samples, tissue fluid, saliva, oral mucosa, nasopharyngeal secretions, body fluids, and urine. Preferably, the biological sample is whole blood, plasma or serum, and more preferably serum.

3. The application of biomarkers as described in claim 2, characterized in that, The expression level of sST2 in the donor's biological sample can be determined by gene / mRNA detection or protein detection.

4. The application of biomarkers as described in claim 2, characterized in that, The biological sample is serum. The diagnostic / risk prediction criteria for srICIAM are that when the sST2 level / concentration in the serum of the donor being tested is above 36.96 ng / mL, it indicates that the donor patient is in a high-risk group for srICIAM. When the serum sST2 level / concentration in the donor is ≥ 36.96 ng / mL and the serum cTnT level / concentration is ≥ 511.0 pg / mL, it indicates that the donor patient has severe myocardial injury and is at risk of developing hormone-resistant ICIs-related myocarditis.

5. The application of biomarkers as described in claim 4, characterized in that, The srICIAM diagnostic / risk prediction criteria are input as a mathematical model into a detection system equipped with a computer or connected to a computer network. The detection system includes an analysis unit and an evaluation unit, wherein the analysis unit is used to detect the levels / concentrations of serum proteins sST2, cTnT, and CK-MB; and the evaluation unit is used to calculate the risk, severity score, or prognosis of srICIAM based on the above protein levels.

6. The application of biomarkers as described in claim 1, characterized in that, The reagents are used to detect the levels of sST2, cTnT, and CK-MB proteins in biological samples based on the following principles: ELISA (enzyme-linked immunosorbent assay), chemiluminescent immunoassay, flow cytometry, Olink proximity extension technology, multiple inflammatory protein detection technology, and Luminex multiplex factor detection technology.

7. The application of biomarkers as described in claim 1, characterized in that, The reagent is a kit, namely an sST2 detection kit for diagnosing hormone-resistant ICIs-related myocarditis; or an sST2+cTnT and / or CK-MB detection kit for diagnosing hormone-resistant ICIs-related myocarditis.

8. The application of biomarkers as described in claim 7, characterized in that, The kit is a protein detection reagent based on the principle of antibody-antigen affinity binding.

9. The application of biomarkers as described in claim 7, characterized in that, The antibody used to recognize sST2 is selected from sST2 monoclonal antibody or sST2 polyclonal antibody; the antibody used to recognize cTnT is selected from cTnT monoclonal antibody or cTnT polyclonal antibody; the antibody used to recognize CK-MB is selected from CK-MB monoclonal antibody or CK-MB polyclonal antibody.

10. The application of biomarkers as described in claim 9, characterized in that, The kit is selected from at least one of the following: latex immunoturbidimetric assay kit, magnetic microparticle chemiluminescence assay kit, radioimmunoassay kit, or immunochromatographic test strip.

Images